Making chemically competent cells Day 1 Grow cells overnight (8-12 hours) Day 2 Before starting Set centrifuge to 4C MgCl2 and CaCl2 solutions Take DMSO out of freezer to thaw Chill microtubes Prepare cells Add 0.5mL of the overnight to 50mL LB Grow until culture is between an OD of 0.2 and 0.4 (check after 1.5hr) Ice for 30 minutes Centrifuge 2700g for 10 minutes at 4C Decant. Dry on paper towel for 1 minute Completely resuspend in 30mL 0.8M MgCl2, 0.2M CaCl2 Gently vortex Centrifuge Decant. Dry on paper towel for 1 minute Fully resuspend in 2mL of 0.1M CaCl2 Add 70ul DMSO while tube is on ice Swirl to mix Ice for 15 minutes Add 70ul DMSO, swirl to mix Dispense 200ul of cells into prechilled microtubes Snap freeze in liquid nitrogen Store at -80c Recommended: Test for efficiency and test for contaminants
